The present invention relates to recombinant DNA which encodes a DNA methylase which methylates cytosine residues which are 5' neighbors of guanine. More specifically, it relates to recombinant DNA which encodes the DNA methylase, M.Sss MQ1, obtainable from the Spiroplasma sp. strain MQ1 and to the production of this enzyme from the recombinant DNA.
DNA methylases transfer methyl groups from the universal methyl donor S-adenosyl methionine to specific sites on the DNA. Several biological functions have been attributed to the methylated bases in DNA. The most established biological function is the protection of the DNA from digestion by cognate restriction enzymes. The restriction modification phenomenon has, so far, been observed only in bacteria. Mammalian cells, however, possess a different methylase that methylates exclusively cytosine residues on the DNA, that are 5' neighbors of guanine (CpG). This methylation has been shown, by several lines of evidence to play a role in gene activity, cell differentiation, tumorigenesis, X-chromosome inactivation, genomic imprinting and other major biological processes. (Razin, A., Cedar, H., and Riggs, R. D. eds. in DNA Methylation Biochemistry and Biological Significance, Springer-Verlag, New York, 1984).
Attempts to clone the mouse DNA methylase resulted in the cloning of full size cDNA of the mouse gene (Bestor, et. al. J. Mol. Biol. 203. 971, 1988). However, there is no evidence that strains containing the cloned gene produce the active enzyme. It should be noted also that the mammalian methylase is a "maintenance" rather than a "de novo" methylase, methylating 10-100 fold more efficiently DNA that is methylated on one of its strands as compared with unmethylated DNA (Razin and Szyf, Biochim Biophys Acta 782, 331, 1984), and is therefore, inefficient in methylating non-methylated DNA (de novo methylation) in vitro. In light of all the above a "de novo" methylating enzyme that methylates exclusively CpG sequences is in demand. Strains of bacteria that would simplify the task of purification as well as providing the means for production of the enzyme in commercial amounts would be useful.
One such methylase, the M.Sss MQ1 DNA methylase methylates exclusively CpG sequences in the DNA (Nur et al, J. Bacteriol 164, 19, 1985) thus mimicing mammalian DNA methylases. One common use of a methylase is to methylate isolated genes. This methylation renders the gene biologically inactive. Currently available methylases methylate only subsets of CpG sequences. For example, M. HpaII methylates the inner cytosine residue of the sequence CCGG, and M. HhaI methylase methylates the inner cytosine residue of GCGC. In contrast, M.Sss MQ1 methylates all CpGs in non-methylated DNA (de novo methylation).
The Spiroplasma sp. strain MQ1 is a member of the Spiroplasmatacea family which belongs to the class Mollicutes of wall-less bacteria. This bacterium is a plant parasite which grow very slowly in culture and reaches a very low cell density (at confluency 10.sup.8 cells/ml). Also the culture medium is relatively complex and expensive. There is, therefore, a commercial incentive to obtain strains of bacteria through recombinant DNA techniques that synthesize this methylase in abundance.